Due to the low abundance of most information-rich bio/chemical species to be detected, the amplification/preconcentration of such species has become the most important issues in bioanalytical research and industry. Considerable improvement in the non-genomic preconcentration methods have been achieved in the last decade, leading at least 100-1000 fold accumulation in sensitivity. Available methods include physical filtration and various novel approaches were studied such as field-amplified sample stacking (PASS), field-amplified sample injection (FASI), isotachophoretic stacking (ITP) and liquid-liquid extraction (LLE). Million-fold amplification of peptides and proteins using micro- and nano-channel hybridized system has been reported. Such methods utilized novel concentration polarization (CP) phenomena near nanochannels where the sample can stay and be accumulated at the ion depletion boundary which was created by CP. However, micro/nanochannels are usually fabricated within the material boundaries confining the flow of the sample. Due to the extremely high concentration gradient after preconcentration procedure, diffusion and dispersion inside the microchannel are critical, limiting the delivery of such high concentrated sample to a desired location such as mass spectrometry (MS) and matrix-assisted laser desorption/ionization-MS (MALDI-MS) systems. In this manner, dispersionless extraction of the concentrated plug has been an important issue. Electrodiffusion, which is proportional to the concentration gradient, or dispersion, which is proportional to the length it travels through, can be controlled only by having less travel length and time from the point of concentration to the point of detection. In this manner, many researchers tried detecting samples inside the channel rather than taking it out, but this limits the usage of the concentration device.
Many efforts have been made for carrying immunoassay inside microchannels in order to utilize the electrokinetic trapping preconcentration. However, implementing immunoassay within a microchannel is generally challenging, especially due to issues related to surface chemistry to immobilize antibodies and other chemical reagents required for immunoassays. On the other hand, conventional immunoassay processes (such as ELISA) were done on open surfaces, with much more robust control on surface chemistry and easier detection. To enjoy both advantages of conventional open surface immunoassays and efficient sample concentration by nanofluidic preconcentration, a preconcentration process outside the microchannel is highly desired.